Although approximately 40% of the risk for coronary heart disease (CHD) can be attributed to genetic variation, we currently understand only a fraction of genetic variants that contribute to this risk. Recent genome wide association studies (GWAS) have pointed to CHD-associated loci that could be contributing to CHD pathobiology via completely unexpected mechanisms, raising the possibility of new therapeutic strategies. Our group has recently shown that one of these CHD-associated polymorphisms directly affects expression of the TCF21 gene, implicating it in CHD pathobiology. TCF21 is a basic helix-loop-helix transcription factor that is found in the developing coronary vasculature, where it plays a critical role in determining cell fate decisions in precursor smooth muscle cells (SMCs). Tcf21-expressing cells are present in the adventitia of human and murine coronary arteries and the aortic media. In response to atherosclerotic disease in the ApoE-/- mouse model, these Tcf21-expressing cells migrate into the developing lesion and align at the fibrous cap, where they show evidence of SMC differentiation. This is especially intriguing given that SMCs are thought to strengthen the fibrous cap structure, which protects against plaque rupture and myocardial infarction. However, the role of Tcf21 in these precursor SMCs is unknown. The series of experiments described herein aim to determine how Tcf21 modulates the phenotype of precursor SMCs and lesion composition in vivo, and to elucidate the molecular pathways by which these effects are mediated. Together, these studies will significantly expand our understanding of the role of SMCs in CHD pathobiology. In Aim 1, we will use conditional Tcf21 knockout and TCF21 transgenic over-expressing Apo E-/- mice to determine how changes in Tcf21 expression affect precursor SMC recruitment and phenotype within the atherosclerotic plaque, and how these changes affect plaque architecture. We will use classical histology and immunohistochemistry to examine plaque composition and structure, with a special focus on the protective fibrous cap. We will track Tcf21-expressing cells via an inducible fluorescent reporter gene. In Aim 2, we will use cells isolated from Tcf21 reporter mice to characterize the in vivo molecular phenotype of Tcf21 lineage-traced cells. A Tcf21-specific fluorescent reporter gene will permanently label Tcf21-expressing cells and allow their identification at various time points during disease progression. These Tcf21 lineage-traced cells will be isolated from aortic root lesions using laser capture microdissection and will undergo RNA sequencing, which will produce a readout of cellular gene expression at progressive stages during plaque development. This will allow molecular phenotyping of these cells and will shed light on important pathways that shape the developing vulnerable atherosclerotic plaque.